Bacteria counter

ABSTRACT

An automated device for detecting measuring and counting growing microcolonies of bacteria by their light-scattering property. The growing bacteria are contained in glass capillary tubes filled with nutrient agar and are counted by passing the capillary tubes through a narrow beam of light. The tubes are mounted in a holder which is, in turn, detachably secured on a carriage. The carriage is translated in a direction lengthwise of the tubes and then sequentially moved transversely in a step equal to the distance between adjacent tubes. Transverse stepping takes place at the end of the longitudinal travel of each tube. A fixed light source and associated stationary optical means projects a narrow light beam which intersects the tubes as they translate. The presence of growing bacterial microcolonies causes light scattering which is detected in the form of pulses of light by a photomultiplier tube located opposite the path of translation of the tubes relative to the light source. The corresponding signal pulses produced by the photomultiplier tube are counted by an electronic counting circuit which provides a fast count of the particles scanned.

United States Patent [72] Inventors Horton E.Dorrnan; 8/1969 Vitt et356/l02X Primary Examiner-Ronald L. Wibert Assistant Examiner-ConradClark Attorney-Herman L. Gordon Rodriguez; Eric W. Youngquist, both ofSilver Spring, all of Md. PP 771,911 ABSTRACT: An automated device fordetecting measuring Med and counting growing microcolonies of bacteriaby their lightscattering property. The growing bacteria are contained inglass capillary tubes filled with nutrient agar and are counted bypassing the capillary tubes through a narrow beam of light. The tubesare mounted in a holder which is, in turn, detachably secured on acarriage. The carriage is translated in a direction lengthwise of thetubes and then sequentially moved transversely in a step equal to thedistance between ad- 356/103, jacent tubes. Transverse stepping takesplace at the end of the 356/167 longitudinal travel of each tube. Afixed light source and as- Charles Soodak, both of Silver Spring;Herbert M. Cullis, College Park; C. David Miller, Greenbelt; RodolfoRamiro [22] Oct. 30, 1968 [45} Patented Dec. 14, 1971 [73] AssigneeBaxter Laboratories, Inc.

Morton Grove, Ill.

[54] BACTERIA COUNTER 21 Claims, 6 Drawing Figs.

sociated stationary optical means projects a narrow light beam whichintersects the tubes as they translate. The presence of growingbacterial microcolonies causes light scattering which m w NHW n os D55nl l 10 t W m m6 4 m m m m4 "4 m2 m m9 m h a C r n 8 u e H S r a C d t dI F l. l. l. 0 5 5 l l is detected in the form of pulses of light by aphotomultiplier tube located opposite the path of translation of thetubes rela- Reierences Cited UNITED STATES PATENTS tive to the lightsource. The corresponding signal pulses produced by the photomultipliertube are counted by an electronic counting circuit which provides a fastcount of the particles scanned.

3/1958 Jones........................... 356/244 BACTERIA COUNTER Thisinvention relates to bacteria counting devices, and more particularly toan apparatus for scanning and counting bacterial microcolonies containedin capillary tubes or equivalent elongated containers.

A main object of the invention is to provide a novel and improvedbacteria counting device which is relatively simple in construction,which is reliable in operation, and which makes it possible to assayviable micro-organisms rapidly and to determine their sensitivity tospecific antibiotics or other materials.

A further object of the invention is to provide an improved automaticscanning, counting and memory system which can be employed to identifyand measure proliferation of bacteria after relatively brief periods ofincubation, the system involving relatively inexpensive components,being relatively compact in size, and eliminating many of thetime-consuming, repetitious and tedious steps involved in conventionalclinical laboratory assay procedures.

A still further object of the invention is to provide an improvedautomated bacteria counting apparatus wherein micro-organisms arenarrowly arrayed in agar culture mediums in transparent capillarycontainers and are exposed to a beam of light which produces lightscatter pulses corresponding to growing colonies of the micro-organisms,the apparatus being arranged to sequentially rapidly translate thecontainers in paths intersecting the light beam, to detect the lightscatter pulses, and to accurately count the pulses electronically.

Further objects and advantages of the invention will become apparentfrom the following description and claims, and from the accompanyingdrawings, wherein:

HO. 1 is a perspective view, partly diagrammatic, of a transportmechanism for moving capillary tubes containing bacteria colonies, asemployed in an apparatus constructed in accordance with the presentinvention.

FIG. 2 is an enlarged vertical cross-sectional view taken substantiallyon line 2-2 of FIG. 1.

FIG. 3 is a fragmentary vertical cross-sectional view takensubstantially on line 3-3 of FIG. 2.

FIG. 4 is a fragmentary vertical cross-sectional view takensubstantially on line 4--4 of FIG. 2.

FIG. 5 is an electrical wiring diagram showing the motor control circuitof the transport mechanism of FIGS. 1 to 4.

FIG. 6 is a block diagram of a counting circuit employed with thetransport mechanism of FIGS. 1 to 5 and forming part of an improvedbacteria counting apparatus according to the present invention.

A photometric method of assaying bacteria by counting light scatterpulses from developing colonies in a capillary tube is described in arecent article by Robert L. Bowman, Philip Blume and Gerald G. Vurekappearing in Science," Vol. 158, Oct. 6, I967, entitled Capillary-TubeScanner for Mechanized Microbiology." This article describes ageneralized procedure, performed manually, wherein a capillary tube 11(see FIG. 6) containing developing bacterial colonies in agar along withvarious nutrients is translated in the path of a narrow light beam 12from a light source 13. The light is focused by a suitable opticalsystem 14. The presence of proliferating bacterial colonies in thecapillary tube causes scattering of the beam, and scattered rays in abeam 15 pass through a microscope objective l6 and are directed therebyto a photomultiplier tube 17 through a slit 18 located at the imageplane of the microscope objective 16. Thus, the photomultiplier tube 17sees" the microcolonies in the form of pulses of scattered light as thecapillary tube 11 translates longitudinally through the incident beam12. This general technique is employed in the apparatus of the presentinvention.

The above-mentioned article points out the desirability of an automatedmechanism for scanning the capillary tubes and for efficiently countingthe photomultiplier signal pulses when their amplitude exceeds apredetermined threshold. Said article suggests the use of a synchronousmotor-driven carriage to translate the capillary tube through theincident light beam and the recording of the scatter pulses on a chart,whereby the recorded charts or accumulated counts of successive scans ofa capillary tube could be compared directly. The article mentions thesue of a plurality of holders supporting respective capillary tubes, theholders being designed to be replaced (manually) precisely on thecarriage without disturbing the orientation of the capillary tubes intheir holders.

It is an aim of the present invention to provide a means ofautomatically shifting the next capillary tube into scanning position asscanning of a preceding tube is completed and to provide a cycle whereina relatively large number of capillary tubes are successively scanned.It is a further important aim of the present invention to provide ameans of automatically digitally recording the total count of thescatter pulses.

The above-described technique and the apparatus of the present inventionfacilitates early and more successful therapy in cases of acutebacterial infection by making it possible to assay viablemicro-organisms rapidly and to determine their sensitivity to specificantibiotics. The apparatus can be used to identify proliferation ofbacteria after relatively brief periods of incubation without theheretofore employed time-consuming, repetitious and tedious steps, suchas those involved where petri dishes are employed as culture vessels andthe microcolonies are developed in planar array on the surfaces of thepetri dishes.

The above technique and the apparatus of the present invention offer away to greatly reduce the time previously required for physicians toobtain biological data necessary to determine and begin optimaltreatment for individual patients. For example, specimens for assay arediluted with measured amounts of melted agar and introduced intocapillary tubes, which are then sealed, placed in a holder, mounted on ascanning carriage, and then moved sequentially through the intersectinglight beam. All colonies or particulate debris within the capillarytubes act as scattering points to produce light scatter pulses. Thesepulses are processed through a suitable amplifier system which deliversthe accepted pulses corresponding to colony counts to an electronicpulse counter.

Since individual bacteria colonies retain their positions in the agar,only those organisms that show growth will produce increased lightscatter pluses when rescanned after incubation. By adding antibiotics tothe agar in different samples, proliferation or nonproliferation oforganisms similarly will show resistance or susceptibility to therespective preparations.

Once an initial light scatter record is obtained, the specimen isincubated and a new recording is taken. Repeat scanning demonstratesadditional pulses due to proliferated bacteria,

and makes it possible to identify and subtract nonincreased pulsescaused by inert particles.

Referring to FIGS. 1 to 4 of the drawings, 18 generally designates acapillary tube transport mechanism constructed in accordance with thepresent invention. The mechanism 18 comprises a rectangular carriage l9slidably mounted on a pair of parallel stationary rails 20 and 21 fixedto a suitable supporting structure, not shown. Thus, the carriage 19 maybe provided with a pair of depending rear corner blocks 22 which havealigned bores slidably receiving the rear rail 20 and with a dependingfront intermediate block 23 which has a bore slidably receiving thefront rail 21. The carriage 19 is thus slidable in either direction onthe parallel rails 20, 21.

The carriage 19 comprises the horizontal rectangular main wall 24 andthe upstanding opposite sidewalls 25 and 26. Main wall 24 is formed withan aperture 27 located above the lamp 13 and the optical assembly 14(not shown in FIGS. 1 to 4) to allow passage therethrough of thescanning beam 12 of FIG. 6.

Designated at M, is a conventional reversible electric motor having therespective right" and left" operating windings shown diagrammatically atR and L in FIG. 5. Motor M, is mounted on a suitable stationary support,not shown, and is coupled through a conventional electrically controlledclutch C to a pinion gear'28 which is drivingly engaged with a rack bar29 secured on the rear marginal portion of main wall 24 and whichextends parallel to rails 20, 21. When either the R or L motor windingand the-clutch C are simultaneously energized, the carriage 19 is driveneither to the right or left on the rails 20,21, depending on which ofthe motor windings R or L is energized. As will be presently described,the translation of the carriage is limited by the provision ofrespective right and left stationary limit switches S and S which closeat the ends of the respective right and left excursions of the carriageand which act to cause reversal of movement of the carriage. Thus, thenormally open limit switch S is mounted on an adjacent stationaryportion of the associated supporting frame, the operating plunger 30 ofsaid switch being axially aligned with and in the path of movement of apin 31 secured to a lug 32 depending from the left corner block 22, Asimilar pin is provided at the right side of the carriage l9, axiallyaligned with and engageable with the operating plunger 33 of thestationary normally open limit switch S As shown in FIG. 5, the limitswitches S and S, are connected in the energizing circuits of respectiverelays K and K The top rear edge portions of sidewalls 25 and 26 areformed to define reduced upstanding ribs 34,34 which are grooved at 35to define opposing inwardly facing channels. Pivotally and slidablyreceived in said channels are respective pins 36,36 fixed to andprojecting from the opposite ends of a bar 37 which is, in turn,pivotally secured to the rear edge ofa rectangular platelike framemember 38. The sidewalls 25 and 26 are recessed at their forward topedge portions, as shown at 39,39 and are formed thereat with reducedribs 40,40. Said ribs are formed at their intermediate portions withaligned serrations 41 defining seats for at times receiving oppositelyprojecting front comer pins 42,42 provided on the platelike frame member38.

As shown in FIG. 2, bar 37 is pivotally connected at its midpoint to themidpoint ofthe rear edge of member 38 by a pivot pin 69.

Pins 42,42 are rigidly secured in and project from the forward portionsof the respective side edges of member 38, and can be successivelyengaged in aligned pairs of V-shaped notches of the serrations 41,thereby providing positive positioning of member 38 on carriage 19. Aswill be presently explained, this provides positive identical steps oftransverse movement of the capillary tubes, whereby to insure that theincident beam 12 will always intersect a capillary tube for properscanning thereof.

The forward portion of frame member 38 is biased downwardly by laterallyprojecting leaf springs 43,43 secured to the bottom surface of itsforward corner portions, the outer ends of the leaf springs beingslidably received in horizontal guide grooves 44 formed in the sidewalls25 and 26.

The transverse stepping mechanism for the frame member 38 comprisesrespective sideplates 45,45 slidably and pivotally mounted on thesidewalls 25 and 26 and having notches 46 at the forward portions oftheir top edges engageable with the pins 42 to lift them out of one pairof aligned serrations 41 and move them into an adjacent pair of alignedserrations 41. The rear portions of the sideplates 45 are formed withlongitudinal slots 47 through which extend headed pins 48 securedrespectively in the sidewalls 25 and 26, whereby the plates 45 areslidably and pivotally connected to said sidewalls. Respectivecylindrical crank members 49,49 are rotatably mounted in the forwardportions of the sidewalls 25 and 26 and are rigidly secured to theopposite ends of a common indexing drive shaft 50. The crank members 49have pins 51 engaged in apertures in the respective sideplates 45,45,whereby said sideplates are simultaneously translated and oscillatedresponsive to the rotation of shaft 50. The amplitude of suchtranslation and oscillation is sufficient to elevate the pins 42,42 outof one aligned pair of serrations 41 and step them into the next pair ofaligned serrations 41 with each revolution of shaft 50. As shown in FIG.1, indexing shaft 50 drives pins 51 in a clockwise direction. Said shaftis provided at its intermediate portion with a radial-toothed gear 52which is meshingly engaged by a pinion gear 53 carried on the shaft ofan indexing motor M mounted beneath and secured to the main wall 24 ofcarriage 19.

Respective horizontal retaining bars 54,54 are secured in the recesses39,39 overlying the serrations 41 but spaced sufficiently above saidserrations to provide adequate clearance for the stepping of pins 42,42from one pair of aligned serrations to the next pair as above described.

A limit switch S is mounted on the associated carriage plate structurein a position such that its operating element will be engaged by a lug55 secured'to the sideplate member 45 of sidewall 25 and will operatethe switch after approximately 200 of clockwise rotation of its pin 51from its lowermost position shown in FIG. 1. The switch 8,; will bereleased shortly after the pin 51 completes 360 of clockwise rotation.

Designated generally at 56 is a capillary tube holder arranged to bereleasably mounted on the frame member 38 in a precisely establishedposition on said frame member. The holder 56 comprises a flat framehaving front and rear arms 57 and 58 and sidearms 59,59 formed withaligned pairs of serrations or grooves 60 adapted to receive capillarytubes 11 and to position said tubes parallel to each other and to thefront and rear arms 57 and 58. A U-shaped resilient wire ball 61 has itsbight portion 62 pivotally connected by apertured hinge lugs 63,63 tothe rear arm 58. The sidearms 64 are clampingly cngageable with thecapillary tubes 11 positioned in the grooves 60, and are lockinglyretained in spring clips 65,65 mounted on front arm 57.

The holder 56 is engageable on the member 38 with the ends of the frontand rear arms 57 and 58 received beneath inwardly projecting flanges66,66 provided on upstanding sideplates 67,67 secured on the sidemarginal portions of member 38. Bowed leaf springs 68,68 arerespectively secured to the undersides of flanges 66,66 with theirdepending ends clampingly engageable with the ends of the front and reararms 57 and 58 of the holder 56 when said holder is inserted. The reararm of member 38 is provided with an upstanding stop pin and with apositioning ball 71 spaced forwardly thereof. Similar positioning balls72,72 are provided in the forward comer portions of member 38. Grooves73 are provided in the holder 56 to receive the positioning balls 71 and72, with the rear edge of the holder in abutment with stop pin 70,whereby to constrain the holder 56 to occupy a fixed precise location onthe member 38.

The grooves 73 may comprise respective V-grooves extending perpendicularto and intersecting their adjacent edges. Thus, then the positioningballs 71 and 72 are seated in their corresponding V-grooves, the holder56 is positively positioned in an accurately reproducible position onmember 38. This is necessary in order to insure that the scanning beam12 will properly impinge on each of the tubes 11 carried by any holderinserted on the member 38 without the necessity of otherwise aligning oradjusting the holder when it is inserted.

The front edge of member 38 is notched away at its intermediate portion,as shown at 74, to facilitate grasping the rear arm 57 of holder 56 ininserting or removing the holder.

A cross rod 75 is secured between upstanding studs 76,76 mounted on theforward end portions of sideplates 67,67, said cross rod serving as ahandle for at times manually positioning the transversely movablecarriage member 38.

It will be noted that the pivot pin 69 in cooperation with bar 37, andthe engagement of the springs 43 in the grooves 44, provide athree-point suspension of the member 38 on the main body of the carriageassembly 19.

A normally closed limit switch S is mounted on the associated adjacentfixed structure, not shown, with its operat ing element 77 in the pathof movement of a pin 78 projecting from the upstanding plate 67 carriedon the margin of member 38 adjacent sidewall 26. As will be presentlydescribed, pin 78 engages element 77 at the end of a run, and therebyopens s-witch S,, which deactivates the mechanism.

Another limit switch S is likewise suitably mounted with its operatinglever 79 in a position to be engaged by a portion of member 35, forexample, the rear end of the right flange 66, when the transportmechanism 18 is in starting position. In starting position the switch Sis closed and the mechanism can be started, as will be presentlydescribed. Starting cannot take place unless switch S, is closed.

In the starting position, namely, with switch S closed, the beam 12 willimpinge on the left end portion of the foremost capillary tube carriedon the holder 56 seated on member 38. At the end of the run, namely withswitch S, open, the beam 12 will impinge on the left end portion of therearmost capillary tube 11 mounted on the holder.

The various switch-operating pins 78, 31, etc., are threadedly engagedwith their supports, so that they can be readily adjusted to provideproper timing of the switching actions performed thereby.

As shown in FIG. 5, the control circuit for the motors M,, M and clutchC includes the various limit switches S,. 5,, S S and S,,, a two-polemanually operated pushbutton switch S,,, and respective relays K,, K Kand K.,

Indexing limit switch S is of the three-pole, double-throw type, whosepoles normally engage their associated upper contacts, as shown.

Again referring to FIG. 5, it will be seen that the R and L windings ofthe scanning motor M, have a common junction, andsaid common junction isconnected to the AC supply wire 9. The remaining terminal of the Lwinding is connected by a wire 81, a bottom contact 82 and theassociated pole 83 of the manually deenergized relay K,,, and a wire 84to an upper contact 85 of the normally deenergized, relay K,,. Theassociated pole 86 is connected to a. wire 87, which is in turnconnected through the normally open contacts 88-89 of the normallydeenergized relay K, to the other supply wire 90 connected to a llO-volt AC source. Thus, the L winding will be energized responsive tothe simultaneous energization of relays K, and K,.

The remaining terminal of the R winding is connected by a wire 91 abottom contact 92 and the associated pole 93 of the normally deenergizedrelay K,, and a wire 94 to the bottom contact 95 of relay K,, normallyengaged by pole 86. Thus, the R winding will be energized responsive tothe energization only of relay K,, with relays K and K deenergized.

One terminal of indexing motor M is connected to wire 9. The otherterminal thereof is connected to a wire 96. Wire 96 is connected to theupper contact 97 of relay K associated with pole 93, to the uppercontact 98 of relay K,, associated with pole 83, and to the lowercontact 99 associated with a pole 100 of the three-pole indexing limitswitch S,. In their normal positions, the respective poles 100, 101 and102 of switch S engage their upper contacts, as shown. The upper contact103 associated with pole 102 is grounded. Pole 102 is connected by awire 104 to a pole 105 of relay K,, and by a further wire 106 to a pole107 of relay K,,, normally engaging a grounded lower contact 108.

Clutch C has one terminal connected to wire 9. The other terminalthereof is connected to a wire 109, which in turn is connected to alower contact 110 of relay K normally engaged by a pole 111, and to alower contact 112 of relay K normally engaged by a pole 113. Pole 111 ofrelay K is connected by a wire 114 to a lower contact 115 of relay K,normally engaged by a pole 116, which is connected to wire 87. Pole 113of relay K is connected by a wire 117 to the upper contact 118 of relayK,, associated with pole 116.

One terminal of the winding of each of relays K,, K,, K,, and K,, isconnected to the negative l2-volt DC supply wire 119. The remainingterminal of the winding of relay K, is connected to a wire 120. Wire 120is connected through the upper contacts 121-122 of relay K, and normallyclosed limit switch 5, to ground. Also, wire 120 is connected throughthe upper contacts 123-124 and pole 125 of a two-pole pushbuttonoperated switch S and normally open limit switch S to ground.

The remaining terminal of the winding of relay K is connected to groundterminal the normally open right-end limit switch S,. The remainingterminal of the winding relay K,, is connected to ground through theleft-end limit switch S,..

The remaining terminal of the winding of relay K, is connected by a wire126 to the upper contact 127 of relay K, associated with pole 105, andsaid wire 126 is connected by a further wire 128 to a wire 129, which isconnected to ground through the lower contacts 130-131 and pole 132 ofpushbutton switch S Wire 129 is also connected to a pole 101 of indexingswitch S,. Pole 101 is engageable with a contact 133, which is connectedby a wire 134 to the lower pole 135 of relay K When relay K, isenergized, pole 135 is engageable with a grounded upper contact 136.

In operation of the circuit of FIG. 5, let it be assumed that a holder56 carrying 12 capillary tubes 11 has been loaded on the transportmechanism 18 and that the carriage 19 is in its starting position andthat the transversely movable transport member 38 is in its rearmostposition, namely, with the pins 42,42 in the rearmost pair of alignedserrations 41. Under these conditions, switch S, is closed. Whenpushbutton switch S is actuated, relay K, becomes energized by a circuitcomprising negative supply wire 119, the winding of relay K,, wire 120,switch contacts 124-125-123, switch 8, and ground. Relay K, closes itsholding contacts 121-122, thereby connecting wire 120 to ground throughnormally closed limit switch S,. Relay K, thus remains energized untilthe end of the run.

The closure of contacts 130-132-131 of switch S, energizes relay K,through a circuit comprising negative supply wire 119, the winding ofrelay K,, wire 128, wire 129, contacts 130-132-31 and ground. Theholding contacts -127 of relay K,, close and keep wire 126 groundedthrough wire 106 and contacts 107-108 of relay K,, as long as said relayK is deenergized.

Clutch C is energized through a circuit comprising line wire 90,contacts 88-89 of relay K,,wire 87, contacts 116-118 of relay K wire117, contacts 113-112 ofrelay K,,, wire 109, the clutch winding, andwire 9.

The L winding of motor M, is energized through a circuit comprising linewire 90, contacts 88-89, wire 87, contacts 86-85 of relay K,, wire 84,contacts 83-82 of relay K,,, wire 81, winding L and wire 9. The carriage19 is then moved to the left by motor M,, releasing switch 8,.

At the end of the leftward scan of the first (foremost) capillary tube11, limit switch S closes and energizes relay K,, by a circuitcomprising negative supply wire 119, a wire 137, the winding of relayK,,, switch S and ground. Clutch C is deenergized by the opening ofcontacts 113-112 and the L motor winding is deenergized by the openingof contacts 83-82. The holding circuit of relay K,, is retained throughwire 104 and contacts 102-103 ofswitch S Indexing motor M is energizedthrough a circuit comprising line wire 90, relay contacts 88-89, wire87, contacts 86-85 of relay K,, wire 84, contacts 83-98 of relay K,,, awire 138, wire 96, the winding of motor M and wire 9.

Motor M rotates shaft 50 clockwise, as above described, and afterapproximately 200 of rotation of pins 51 (by which time the pins 42,42have been transferred forwardly to the next pair of aligned serrations41,41) lug 55 actuates limit switch S,. This deenergizes relay K,, bythe opening of contacts 102-103, but motor M continues to run, sincecontacts 100-99 remain closed, until shortly after 360 of rotation ofshaft 50, whereupon contacts 100-99 open and deenergize motor M Thedeenergization of relay K,, energizes the R winding of motor M, by acircuit comprising line wire 90, relay contacts 88-89, wire 87, contacts86-95 of relay K,, wire 94, contacts 93-92 of relay K,, wire 91, the Rwinding, and wire 9. Clutch C is likewise energized by the closure ofcontacts -116 of relay K,, by a circuit comprising wire 90, contacts88-89, wire 87, contacts 116-115, wire 114, contacts 111-110 of relayK,, a wire 139, the clutch winding, and wire 9. Motor M, thus movescarriage 19 rightwards, with the second capillary tube 1 1 in the pathof scanning beam 12.

At the right end of the scanning travel forward step, during carriage19, limit switch S is closed, energizing relay K through a circuitcomprising negative supply wire 119, wire 137, the winding of relay Kswitch S and ground. This opens contacts 111-110 and 92-93, deenergizingmotor M Indexing motor M is again energized by the closure of relaycontacts 93-97, by a circuit comprising line wire 90, contacts 88-89,wire 87, contacts 86-95 of relay K wire 94, contacts 93-97 of relay Kwire 96, the winding of motor M and wire 9. Another indexing cycleoccurs, moving the member 38 through a second forward step, during whichrelay K becomes energized through a circuit comprising negative supplywire 119, the winding of relay K wire 128, wire 129, contacts 101-133 ofswitch S wire 134, contacts 135-136 of relay K and ground. Relay K isheld energized by the holding circuit including wire 126, contacts105-127, wire 106, contacts 107-108 of relay K and ground.

Clutch C is then energized through contacts 116-118 of relay K and the Lwinding of motor M, is likewise energized through contacts 86-85 ofrelay K repeating the leftward scan for the third capillary tube 11, asabove described for the first capillary tube. The action continues untilthe completion of the twelfth scan, wherein the carriage 19 completesthe last rightward scan, whereby pin 78 opens switch S and releasesrelay K,. This opens contacts 88-89 and deactivates the mechanism.

It is thus seen that after a holder 56 carrying the full complement of12 capillary tubes has been placed in the carriage member 38 as abovedescribed, with the parts in starting position, actuation of pushbuttonswitch S will begin a cycle of operation wherein the 12 capillary tubes11 are individually linearly scanned at the same rate, and that thecycle will terminate after the 12th tube has been scanned, with theholder 56 in its foremost position, namely, with the pins 42,42 restingin the foremost pair of aligned serrations 41,41. The holder 56 may thenbe removed, a new holder may be inserted, the member 38 may be manuallyreturned to its starting position, and the capillary tubes 11 of the newholder may then be scanned by again actuating pushbutton switch S Thelight scatter pulses produced during the scanning of each tube 11 causea corresponding series of signal pulses to be produced by thephotomultiplier tube 17 (FIG. 6)-. The pulses of each scan are countedand recorded by the counting circuit diagrammatically shown in FIG. 6.To initiate counting action at the end of each scan, respective pairs ofcontacts 141-142 and 143-144 of relays K and K close, these contactsbeing connected in parallel in a switching circuit controlling a gate Gwhich, in turn, controls the action of the printer and oscillator ofFIG. 6, as will be presently described. Thus, as shown in FIG. 5, therespective pairs of contacts 141-142 and 143-144 are connected inparallel to the control wires 145 and 146 leading to gate G whereby saidgate is turned on responsive to the closure of either left-end limitswitch S or right-end limit switch 8,, which occurs at the end of eachtube scan.

The signals from the photomultiplier tube 17 are delivered (aconventional band-pass amplifier A and emerge as pulses corresponding tocounts. The count pulses are tallied from one to 999 by a decade counterD D D This decade counter is of the BCD type, and thus there are fouroutputs for each decade. Each output is individually cornpared to thecorresponding output of decade counter D -D -D by gates G G mG If anyoutput is different from the corresponding output with which it iscompared, then the gate G and G or...G, which compares them, places asignal into gate G or G or 0, (as is appropriate), allowing a signalfrom the oscillator A to enter the appropriate decade counter D, or D orD and simultaneously advancing count wheel W or W or W (as isappropriate) within the printer. Thus, the count wheel will advance tothe numeral appropriate for its decade, at which time oscillator pulseswill become blocked by the action of gate G or G or G (as isappropriate), each of which will turn off when each of its four inputsare off.

Oscillator A is a multivibrator producing square waves of frequencyappropriate to the speed capabilities of the print wheels. A, isfurthermore activated by gate G which allows oscillations only at theend of the scan of each tube, while no further counts are beinggenerated by amplifier A,.

When all three count wheels have reached their appropriate numerals, andtherefore gates G G and G are all off (in the zero state), then gate Gturns on, and if gate G is also on, this causes gate G to turn on and tocause the printer to print" and to reset decade counters D D,...D tozero.

Thus, assume that in normal operation amplifier A delivers, for example,238 pulses for the scan of some given tube. Then D, has the output1,000, D: has the output 001 l, and D;, has the output 0010 in binarycounting. At the end of the scan, gate G turns on, starting oscillator Aproducing a square wave at, say 30 c.p.s. Gate G allows this signal toenter gate D and the "units" count wheel system, until D reads 1,000, atwhich time the units wheel reads eight." At this point, gate G turnsoff. The 10's and l00s" wheels operate similarly. When gates G G and Gare all off," and gate G is on, gate G causes the printer to print andalso causes all the D units to clear to zero.

While a specific embodiment of an automatic bacteria counter has beendisclosed in the foregoing description, it will be understood thatvarious modifications within the spirit of the invention may occur tothose skilled in the art. Therefore it is intended that no limitationsbe placed on the invention except as defined by the scope of theappended claims.

What is claimed is:

1. In a particle counting apparatus comprising a fixed light sourceproviding a directed scanning beam; a carriage member; means on saidcarriage member defining a plurality of substantially parallel spacedelongated transparent sample receptacles located so that one of saidreceptacles is in the path of said scanning beam; means for moving saidcarriage member longitudinally along the axes of said receptaclesthereby to scan each one of said receptacles in one direction to apredetermined carriage position; said moving means comprising a supportmovably mounting said carriage, fixed guide means extending parallel tosaid receptacles, means mounting said support on said guide means so asto be constrained to move there along, and means to move said support onsaid fixed guide means; light-sensitive means located to receive lightscattered from particles in said one receptacle as a result of saidscanning action; means to move said carriage member transversely througha distance substantially equal to the spacing between adjacent samplereceptacles responsive to said predetermined longitudinal positioning ofsaid carriage member thereby to dispose the receptacle next adjacent tosaid one receptacle to the path of said scanning beam; and means to thenmove said carriage member longitudinally in a reverse direction wherebysaid next adjacent receptacle is scanned in said reverse direction.

2. The particle counting apparatus of claim 1 wherein said predeterminedlongitudinal carriage member position is defined by a location of saidcarriage member wherein the path of said scanning beam intersects an endportion of said one receptacle whereby upon transverse movement of saidcarriage member said next adjacent receptacle starting at itscorresponding end portion is scanned to its other end.

3. The particle counting apparatus of claim 2, wherein means areprovided to again move the carriage member transversely through anadditional distance substantially equal to the spacing between adjacentreceptacles responsive to the reverse movement of the carriage member toa location wherein the scanning beam intersects an opposite end portionof said next adjacent receptacle, and means to then again move thecarriage member in the first-named longitudinal direction.

4. The particle counting apparatus of claim 3 comprising means todeactivate the carriage-moving means responsive to the completion of thelongitudinal scanning movement of the last of the receptacles throughthe scanning beam.

5. The particle counting apparatus of claim 1 wherein saidlight-sensitive means includes an electronic device generating anelectrical signal responsive to light received thereby, and meanscounting the electrical signals generated by the light scattered fromthe respective particles during said scanning action.

6. The particle counting apparatus of claim 5 wherein said countingmeans includes means to tally said count for said scanning action indigital form.

7. The particle counting apparatus of claim 6, wherein said countingmeans further includes means to print out said count and means to resetsaid counting means to zero, and means to simultaneously energize saidprintout means and said reset means responsive to said predeterminedlongitudinal positioning of the carriage member.

8. The particle counting apparatus of claim 1 wherein said carriagemember is provided with means defining a seat, said receptacle-definingmeans comprising a holder removably mounted on said seat, said samplereceptacles comprising individual transparent tubes, said holder havingpositioning means engaged with the tubes to lock them in spaced parallelfixed longitudinal positions on said holder, and interengagingpositioning means on said holder and said seat constraining said holderto occupy a fixed position on said carriage member.

9. The particle counting apparatus of claim 1 wherein said supportincludes transversely extending guide means, and means on the carriagemember engaged with said transversely extending guide means andconstraining the carriage member to move therealong.

10. The particle counting apparatus of claim 9 comprising meansproviding a sliding and pivotal connection of said carriage member withsaid transversely extending guide means, said pivotal connection beingon an axis parallel to said receptacles.

11. The particle counting apparatus of claim 1 wherein said means tomove said carriage member transversely through a distance substantiallyequal to the spacing between adjacent receptacles comprises crank meanson said support rotating on an axis parallel to said receptacles, andinterlocking means on said crank means and said carriage member forstepping said carriage member through said distance between adjacentreceptacles responsive to each revolution of said crank means.

12. The particle counting apparatus of claim 11 wherein saidinterlocking means comprises a transverse arm, means slidably andpivotally connecting said arm to said support, means drivingly andpivotally connecting said arm to said crank means whereby said arm istranslated and oscillated responsive to rotation of said crank means, alateral projection on said carriage member, and means on said armdrivingly engageable with said projection during a portion of thetranslation and oscillation ofsaid arm.

13. The particle counting apparatus of claim 12, wherein said arm isprovided at its top edge with a series of drive notches in which saidprojection is sequentially receivable responsive to successiverevolutions of said crank means.

14. The particle counting apparatus of claim 13, wherein said supporthas a transverse wall element underlying said projection, the top edgeof said wall element being formed with a series of serrations spacedapart substantially the same distance as said receptacles, saidprojection being receivable successively between adjacent serrations toprovide registry of a receptacle with the scanning beam when saidreceptacle is stepped into scanning position.

15. The particle counting apparatus of claim 11, wherein said crankmeans is provided with a driving shaft and an indexing electric motordrivingly connected to said driving shaft.

16. The particle counting apparatus of claim 15, wherein said indexingmotor has an energizing circuit including switch means closingresponsive to said predetermined longitudinal positioning of saidcarriage member.

17. The particle counting apparatus of claim 16, wherein said means tomove said support on said fixed guide means comprises a reversibleelectric motor drivingly coupled to said support, said motor havingrespective forward and reverse windings, circuit means energizing one ofsaid windings during the scanning movement in one direction of saidsupport, circuit means deenergizing said one of the windings during theoperation of said indexing motor, circuit means terminating operation ofsaid indexing motor after the carriage member has been stepped, andcircuit means energizing the other winding upon termination of theoperation of said indexing motor.

18. A holder for use with a scanning and particle counting apparatusincluding a carriage member movable in a plurality of directions, saidholder comprising a substantially planar body, a multiplicity ofelongated transparent sample tubes disposed substantially adjacent oneanother in side-by-side parallel relation on said body, means on saidbody surface opposed from said sample tubes adapted for removablemounting cooperation with said carriage member whereby said body ispredeterminately positioned thereon so that said tubes are individuallyand sequentially scanned during carriage movement.

19. The holder of claim -18 wherein said sample tubes are removablyreceived by said holder, and means on said holder cooperating with saidsample tubes to constrain movement of the same in directions both alongand normal to their axes when received on said holder.

20. The holder of claim 19 wherein said holder body provides a centralopening bounded on opposite sides by a pair of arms, said cooperatingmeans including a multiplicity of equidistantly spaced grooves formed insaid pair of arms, one groove in each arm providing support for oppositeends of said sample tubes on an upper surface of said holder.

21. The holder of claim 20 wherein said cooperating means additionallycomprises a resilient clamp element, said clamp element beingsubstantially C-shaped and pivotally mounted at its bight portion to arear arm of said holder, the sides of said clamp element positioned tooverlie said grooves, and a pair of lugs supported by a front arm ofsaid holder for releasably receiving said clamp element sidearms.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION "Patent No. 3,627, 424 Dated December 14, 1971 In n fl Horton E. Dorman, et a1.

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below: a 1

Abstract, line 1, aifter "detecting" include Column 1, line 9, change"micro-organisms" to microorganisms--.

line 22, change "micro-organisms" to -microorganisms-. line 25, change"micro-organisms" to --microorganisms-. line 50, change "light scatter"to --1ightscatter-.

Column 2, line 4, change "sue" to -use.

line 18, change "micro-organisms" to microorganisms-. line 52, change"nonincrea'sed." to nonincreased-.

Column 3, line 28, change "platelike" to plate like-. 1 line 33, change"platelike" to plate -like- Column 5, line 29, change "manually" ton0rma1ly--.

Column 6, line 31, change "130-132 31" to 130-132 131 Column '7, line 1,delete 'forward step, during" and substitute of.

line 59, delete and and before "a" insert -to--. line 66, change "and"to -or--. I

Signed and sealed this 26th day of September 1972.

(SEAL) Attest:

EDWARD M.FI.ETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissionerof Patents FORM PO-IOSO (10-69) uscoMM-Dc 60376-P6O Ill. GOIIIIIII'IDIIIYIIG OI'I CI 1 "I, O-IOl-Sl

2. The particle counting apparatus of claim 1 wherein said predeterminedlongitudinal carriage member position is defined by a location of saidcarriage member wherein the path of said scanning beam intersects an endportion of said one receptacle whereby upon transverse movement of saidcarriage member said next adjacent receptacle starting at itscorresponding end portion is scanned to its other end.
 3. The particlecounting apparatus of claim 2, wherein means are provided to again movethe carriage member transversely through an additional distancesubstantially equal to the spacing between adjacent receptaclesresponsive to the reverse movement of the carriage member to a locationwherein the scanning beam intersects an opposite end portion of saidnext adjacent receptacle, and means to then again move the carriagemember in the first-named longitudinal direction.
 4. The particlecounting apparatus of claim 3 comprising means to deactivate thecarriage-moving means responsive to the completion of the longitudinalscanning movement of the last of the receptacles through the scanningbeam.
 5. The particle counting apparatus of claim 1 wherein saidlight-sensitive means includes an electronic device generating anelectrical signal responsive to light received thereby, and meanscounting the electrical signals generated by the light scattered fromthe respective particles during said scanning action.
 6. The particlecounting apparatus of claim 5 wherein said counting means includes meansto tally said count for said scanning action in digital form.
 7. Theparticle counting apparatus of claim 6, wherein said counting meansfurther includes means to print out said count and means to reset saidcounting means to zero, and means to simultaneously energize saidprintout means and said reset means responsive to said predeterminedlongitudinal positioning of the carriage member.
 8. The particlecounting apparatus of claim 1 wherein said carriage member is providedwith means defining a seat, said receptacle-defining means comprising aholder removably mounted on said seat, said sample receptaclescomprising individual transparent tubes, said holder having positioningmeans engaged with the tubes to lock them in spaced parallel fixedlongitudinal positions on said holder, and interengaging positioningmeans on said holder and said seat constraining said holder to occupy afixed position on said carriage member.
 9. The particle countingapparatus of claim 1 wherein said support includes transverselyextending guide means, and means on the carriage member engaged withsaid transversely extending guide means and constraining the carriagemember to move therealong.
 10. The particle counting apparatus of claim9 comprising means providing a sliding and pivotal connection of saidcarriage member with said transversely extending guide means, saidpivotal connection being on an axis parallel to said receptacles. 11.The particle counting apparatus of claim 1 wherein said means to movesaid carriage member transversely through a distance substantially equalto the spacing between adjacent receptacles comprises crank means onsaid support rotating on an axis parallel to said receptacles, andinterlocking means on said crank means and said cArriage member forstepping said carriage member through said distance between adjacentreceptacles responsive to each revolution of said crank means.
 12. Theparticle counting apparatus of claim 11 wherein said interlocking meanscomprises a transverse arm, means slidably and pivotally connecting saidarm to said support, means drivingly and pivotally connecting said armto said crank means whereby said arm is translated and oscillatedresponsive to rotation of said crank means, a lateral projection on saidcarriage member, and means on said arm drivingly engageable with saidprojection during a portion of the translation and oscillation of saidarm.
 13. The particle counting apparatus of claim 12, wherein said armis provided at its top edge with a series of drive notches in which saidprojection is sequentially receivable responsive to successiverevolutions of said crank means.
 14. The particle counting apparatus ofclaim 13, wherein said support has a transverse wall element underlyingsaid projection, the top edge of said wall element being formed with aseries of serrations spaced apart substantially the same distance assaid receptacles, said projection being receivable successively betweenadjacent serrations to provide registry of a receptacle with thescanning beam when said receptacle is stepped into scanning position.15. The particle counting apparatus of claim 11, wherein said crankmeans is provided with a driving shaft and an indexing electric motordrivingly connected to said driving shaft.
 16. The particle countingapparatus of claim 15, wherein said indexing motor has an energizingcircuit including switch means closing responsive to said predeterminedlongitudinal positioning of said carriage member.
 17. The particlecounting apparatus of claim 16, wherein said means to move said supporton said fixed guide means comprises a reversible electric motordrivingly coupled to said support, said motor having respective forwardand reverse windings, circuit means energizing one of said windingsduring the scanning movement in one direction of said support, circuitmeans deenergizing said one of the windings during the operation of saidindexing motor, circuit means terminating operation of said indexingmotor after the carriage member has been stepped, and circuit meansenergizing the other winding upon termination of the operation of saidindexing motor.
 18. A holder for use with a scanning and particlecounting apparatus including a carriage member movable in a plurality ofdirections, said holder comprising a substantially planar body, amultiplicity of elongated transparent sample tubes disposedsubstantially adjacent one another in side-by-side parallel relation onsaid body, means on said body surface opposed from said sample tubesadapted for removable mounting cooperation with said carriage memberwhereby said body is predeterminately positioned thereon so that saidtubes are individually and sequentially scanned during carriagemovement.
 19. The holder of claim 18 wherein said sample tubes areremovably received by said holder, and means on said holder cooperatingwith said sample tubes to constrain movement of the same in directionsboth along and normal to their axes when received on said holder. 20.The holder of claim 19 wherein said holder body provides a centralopening bounded on opposite sides by a pair of arms, said cooperatingmeans including a multiplicity of equidistantly spaced grooves formed insaid pair of arms, one groove in each arm providing support for oppositeends of said sample tubes on an upper surface of said holder.
 21. Theholder of claim 20 wherein said cooperating means additionally comprisesa resilient clamp element, said clamp element being substantiallyC-shaped and pivotally mounted at its bight portion to a rear arm ofsaid holder, the sides of said clamp element positioned to overlie saidgrooves, and a pair of lugs supported by a front arm of said holder forreleasably receiving said cLamp element sidearms.